Stress granules are non-membranous assemblies of mRNA and protein (mRNP) that form when translation initiation is limiting, which occurs during many stress responses including glucose starvation, heat stress, osmotic stress, and oxidative stress. Stress granules are thought to influence mRNA function, localization, and to affect signaling pathways. Normally, stress granule formation is a dynamic, reversible process that relies on particular RNA-binding proteins that harbor self-interacting domains of low sequence complexity (LC domains). However, a disturbance in the assembly and/or dynamics of these structures is closely associated with a wide array of human diseases, including cancer, infectious diseases and neurodegenerative diseases such as Alzheimer's, Huntington's, Parkinson's, frontotemporal dementia (FTD), and amyotrophic lateral sclerosis (ALS).
The GTPase-Activating Protein SH3 Domain-Binding Proteins (G3BPs), G3BP1, G3BP2a and G3BP2b, are important regulators of stress granule dynamics. G3BP1 has been reported to play a critical role in the secondary aggregation step of stress granule formation, and has been used as a reliable marker of stress granules. The misregulation of stress granule dynamics has been reported in many forms of ALS. G3BP1 is critical for neuronal survival since G3BP1 null mice demonstrate widespread neuronal cell death in the central nervous system. Although single knockout of either G3BP1 or G3BP2 partially reduces the number of stress granule-positive cells induced under stress conditions, the knockout of both genes eliminates stress granule assembly.
To facilitate the analysis of G3BP function, G3BP1 has been fused to, e.g., Green Fluorescent Protein (GFP). However, G3BP fusion proteins for selectively inducing stress granule formation have not been described. Rather, conventional approaches of using sodium azide, arsenite, osmotic (e.g., sorbitol), hypoxia, and heat shock are disclosed for stimulating stress granule assembly. Notably, these toxic conditions confound studies for assessing the role of stress granules in diseases such as ALS, FTD, and cancer. Therefore, there is a need in the art for a noninvasive method of inducing stress granule formation in cells.